The present invention relates to plasmid vectors containing p185neu-encoding sequences and the use thereof in DNA vaccination against tumours. The plasmids according to the invention contain sequences encoding different fragments of human or rat oncoprotein p185neu and are able to induce a humoral or cell-mediated immune response against tumours expressing oncogenes of the ErbB family.
The invention also relates to pharmaceutical compositions containing said plasmids and their use for the prevention or therapy of p185neu-expressing tumours.
Protein p185neu, one of the most studied tumour antigens, has raised great interest as target for immune therapy against cancer, due to its presence on the cell membrane of some of the most common human carcinomas.
p185neu is a membrane receptor encoded in the rat by proto-oncogene Her-2/neu and belonging to the family of Class I Tyrosine Kinase Receptors (RTKs), which also comprises the Epidermal Growth Factor Receptor EGF-R (ErbB-1) and other receptors related thereto (ErbB-3, ErbB-4). These receptors are involved in cell proliferation and differentiation (Hynes and Stern, 1994 BBA 1198:165) and therefore attract a great biological and clinical interest. The receptor consists of three well distinguished domains: an extracellular, transmembrane and intracytoplasmic domain. p185neu is involved in the complex network of mechanisms of intracellular signal transduction and intracellular communication that regulate proliferation and differentiation processes (Boyle 1992 Curr. Op. Oncol. 4:156). Oncogene neu is named after the chemically-induced rat neuroglioblastoma from which it was first isolated. This activated neu form has a single point mutation that results in the replacement of “A” with “T” and in the consequent substitution of the Valine residue at position 664 of p185neu with a glutamic acid residue (Val664Glu) (Bargmann et al. 1986, Cell 45:649).
Also the human neu homologous, ErbB-2, has been isolated and characterised and it has been demonstrated that both rat HER2/neu receptor and human ErbB2 have a significant homology with EGFR (Coussens et al. 1985, Sciente 230:1132; Yamamoto et al. 1986, Nature 319:230). While a genetic mutation in the rat sequence is the cause of constitutive receptor activation through dimerization, in ErbB-2 positive human tumours an aberrant expression of the oncogene is observed (Di Marco et al. 1990, Mol. Cell. Biol. 10: 3247; Klapper et al., 2000, Adv Cancer Res, 77:25), even though, in rare cases, activating point mutations and abnormal splicing mechanisms have been found (Kwong et al., 1998, Mol Carcinog, 23:62; Xie et al., 2000, J Natl Cancer Inst, 92:412). The overall effect is similar: gene amplification and increase in the transcription level determine an excess of p185neu membrane receptor, with consequent increase of active dimers intracellularly transducing growth signals in a ligand-independent manner. The crystal structure of human and rat p185neu extracellular region recently reported shows that this protein is characterised by a rigid conformation that allows to interact with other ErbB receptors, without directly binding any ligands, and trigger the proliferation signal transduction (Cho H S et al. 2003, Nature 421:756).
Under normal circumstances, human p185neu is involved in organogenesis and epithelial growth; it is expressed at high levels during placenta formation and fetal development, whereas it is present at very low levels in adult tissues (Press et al. 1990, Oncogene 5:953).
Several studies have demonstrated that human p185neu overexpression is associated to the neoplastic process and to the level of tumor aggression. The overexpression of p185neu has been described in lung (Kern et al. 1986, Cancer Res. 50:5184), colon (Cohen et al. 1989, Oncogene 4:81), ovary (Slamon et al. 1989, Science 244:707) adenocarcinomas and in a high number of human mammary carcinomas (Slamon et al. 1989, Science 244:707; Jardines et al. 1993, Pathobiology 61:268).
The fundamental properties that make p185neu an optimal target for plasmid vaccination are: a) its direct involvement in cell growth and carcinogenesis, therefore clone variants that, due to tumour genetic instability, lose the expression of this antigen also lose their tumorigenicity; b) its expression on the plasmatic membrane, which makes it recognizable by antibodies even in tumour cells that lose the expression of the major histocompatibility system (Lollini P. and Forni G. 2003, Trends Immunol. 24: 62).
Studies carried out on mice transgenic for the activated rat oncogene Her-2/neu (which spontaneously develop p185neu positive mammary tumours) and on murine models using p185neu positive transplantable tumour lines, have demonstrated the possibility to prevent and cure preneoplastic lesions. As regards in particular the prevention of mammary tumours in mice transgenic for rat activated Her-2/neu, we have demonstrated that the plasmid coding for the extracellular and transmembrane domains of rat p185neu is able to induce an in vivo protection more effective than the plasmid encoding for the full-length rat p185neu or for the extracellular domain only (secreted antigen) (Amici A. et al. 2000, Gene Ther., 7: 703; Rovero S. et al. 2000, J. of Immunol., 165: 5133). Similar results have been reported by Chen et al. (1998, Cancer Res 58:1965). Other authors have demonstrated that plasmids encoding for p185neu—either unvaried or mutated so as to eliminate its tyrosine-kinase activity—are effective in preventing the onset of tumours following to p185neu-positive cells inoculum (Wei W Z et al. 1999, Int. J. Cancer 81: 748). Moreover, plasmids devoid of the signal responsible for the processing through the endoplasmic reticulum (leader), which determines cytoplasmic localization of p185neu antigen, proved equally effective. The protection induced by different plasmids was mainly mediated by a humoral immune response in the case of membrane expression of p185neu and by a T-lymphocyte-mediated immune-response in the case of cytoplasmic localization (Pilon S A et al. 2001, J. of Immunol. 167: 3201). However, combined vaccination with plasmids inducing p185neu overexpression in both the cytoplasm and the membrane was more effective in protecting against tumour growth (Piechocki M P et al. 2001, J. Immunol. 167: 3367).
Thus, the balance between different immune response mechanisms might be particularly important (Reilly et al., 2001, Cancer Res. 61: 880). Moreover, it has been observed that the vaccination with plasmids encoding for extracellular and transmembrane domains of rat p185neu is able to eradicate tumour masses with 2 mm diameter, upon inolculum of cells overexpressing p185neu, through a number of different effector mechanisms of the immune system (T helper and T killer cells, antibodies, macrophages, neutrophiles, natural killer cells, Fc receptors, gamma interferon and perforins), which cooperate to tumor rejection (Curcio C. et al. 2003, J. Clin. Invest. 111: 1161).